Abstract 2466: Metabolic characterization of breast cancer cells and extracellular vesicles using fluorescence lifetime imaging microscopy
Authors: Elisabeth M. Martin, Janet E. Sorrells, Edita Aksamitiene, Prabuddha Mukherjee, Aneesh Alex, Eric J. Chaney, Marina Marjanovic, Stephen A. Boppart
Journal: Cancer Research (2022)

Abstract Extracellular vesicles (EVs) mediate cellular communication by exchanging molecules such as proteins and nucleic acids. EVs are often generated in large quantities by cancer cells and are considered a promising diagnostic biomarker that may provide information on the tumor microenvironment. Tumor cells display characteristic metabolic changes such as the Warburg effect. However, many methods to characterize the metabolism of cells or EVs are destructive or indiscriminate, losing their individual heterogeneity. There is a need to better characterize the metabolism of individual breast cancer cells and single EVs to provide insights into their heterogeneous nature and interactions in the tumor microenvironment. Two-photon fluorescence lifetime imaging microscopy (FLIM), a label-free metabolic imaging technique that can spatially map the autofluorescence properties of NAD(P)H, was used to metabolically characterize breast cancer cells and their derived EVs. EVs of size 100-1000 nm were extracted via differential ultracentrifugation from media conditioned with breast cells. Metabolic stressors including various concentrations of glucose, pyruvate, and lactate were placed on human breast cancer (MDA-MB-231) cells in culture. Subsequently, cells and EVs were imaged with FLIM to determine the metabolic relationship between individual cells and EVs. Additionally, normal human breast cells (MCF10A) and breast cancer cells (MDA-MB-231, MCF7) were imaged with FLIM to determine the metabolic signatures of the three breast cell lines. Images were analyzed using a custom EV detection algorithm, FLIM-phasor analysis, and CellPose. EV collection was validated with Nanotracker Analysis, transmission electron microscopy, and Raman spectroscopy. Human MCF10A cell populations showed significantly lower mean lifetimes than either MDA-MB-231 or MCF7 cells. This suggests that FLIM can be used to differentiate populations of cancerous and non-cancerous breast cells in an individual, non-invasive manner. MDA-MB-231 EVs showed a Gaussian and heterogenous metabolic distribution of NAD(P)H fluorescence lifetimes. This suggests a range of different NAD(P)H species that are free and protein-bound. Fluorescence lifetimes from MDA-MB-231 EVs also demonstrated a wider standard deviation from their corresponding parent cells, and a different distribution from various sub-cellular regions of the parent cell. Additionally, when metabolites available in culture media were changed to induce metabolic changes in cells, similar metabolic changes were reflected in the signatures of their released EVs. These results indicate that FLIM can be a powerful label-free non-perturbative tool to examine both single cells and EVs, as well as their heterogeneous populations, in order to further understand the concurrent metabolic properties of breast cancer cells and their released EVs. Citation Format: Elisabeth M. Martin, Janet E. Sorrells, Edita Aksamitiene, Prabuddha Mukherjee, Aneesh Alex, Eric J. Chaney, Marina Marjanovic, Stephen A. Boppart. Metabolic characterization of breast cancer cells and extracellular vesicles using fluorescence lifetime imaging microscopy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2466.